The US Tox21 Collaboration: Advances Made and Lessons Learned Chairpersons: Linda Birnbaum, NIEHS and NTP Robert Kavlock, US EPA/ORD Monday, March 23, 2015 9:15 am – 12:00 noon CC Ballroom 6E

2004 NTP Vision and Roadmap for the 21st Century 4/23/2017 2004 NTP Vision and Roadmap for the 21st Century To meet the challenges of 21st century toxicology, the 2005 NTP Roadmap included a major initiative to develop a high throughput screening (HTS) program with 3 main goals: To prioritize chemicals for further in-depth toxicological evaluation. To identify mechanisms of toxicity (characterize toxicity pathways, facilitate cross-species extrapolation, provide input to models for low- dose extrapolation). To develop predictive models for in vivo biological response in humans.

Toxicity Testing in the 21st Century 4/23/2017 Toxicity Testing in the 21st Century 2007 National Academy of Science Report Envisions a not-so-distant future in which virtually all routine toxicity testing would be conducted in vitro in human cells or cell lines by evaluating perturbations of cellular responses in a suite of toxicity pathway assays using high throughput robotic assisted methodologies.

Transforming Environmental Health Protection Collins, FS, Gray, GM, Bucher, JR (2008) Transforming Environmental Health Protection. Science 319:906-7.

Formation of the Tox21 Program 5-year Memorandum of Understanding (MoU) on “High- Throughput Screening, Toxicity Pathway Profiling, and Biological Interpretation of Findings”. Released on Feb 14, 2008, signed by NHGRI (F.S. Collins), NIEHS/NTP (S.H. Wilson), and EPA (G.M. Gray). Revised 5-year MoU to add FDA signed on July 19, 2010, by NHGRI (E.D. Green), NIEHS/NTP (L.S. Birnbaum), EPA (P.T. Anastas), and FDA (J. Woodcock). Known informally as Tox21 for Toxicology in the 21st Century.

Purpose of Tox21: Bringing a New Era in Toxicology Support the evolution of toxicology from a mostly observational science to a predominantly predictive science focused upon mechanism-based, biological observations using cultured cells, model tissues, and lower organisms.

Tox21 Goals Research, develop, validate, and translate new chemical testing methods that characterize toxicity pathways. Use new technologies to develop models that can be used to more effectively predict how chemicals will affect biological responses. Gain better understanding of the relationship between chemicals and human diseases, in order to: Prioritize compounds for more comprehensive testing. Identify mechanisms of action. Develop predictive models of human disease.

The US Tox21 Collaboration: Advances Made and Lessons Learned Rusty Thomas, US EPA - The US EPA ToxCast Program: Moving from Data Generation to Application. Anton Simeonov, NCATS - Tox21 Phase II: Testing the 10K Library in Quantitative High-Throughput Screening Assays. Rick Paules, DNTP/NIEHS - Tox21 Phase III: Improving on Biological Coverage, Relevance, and Public Outreach. Warren Casey, DNTP/NIEHS - Prioritization and Predictive Toxicology: Estrogen Receptor Active Compounds. Tina Bahadori, US EPA - From Data to Decisions—An End User’s Perspective.

4/23/2017

Tox21’s Unique Capabilities Unique capabilities not currently available elsewhere: Robotics. High throughput screening platform. Compound handling capabilities. Informatics tools. In the past 2 years, NCATS has been screening more than 10,000 compounds chosen jointly by the NTP, NCATS, and EPA against a set of key nuclear receptors and stress response pathways using cell-based assays.   This screening, still in progress, has greatly advanced our understanding of the potential toxicity of many substances of concern.

Tox21’s Next Phase – Improving Coverage & Relevance Increased use of computational models to predict toxicity and metabolism. Increased focus on human cells with known ability to metabolize chemicals  Expand our understanding of biology by developing and implementing a high throughput and low cost approach to measure the expression level of all genes in a cell at once – Toxicogenomics Increase use of stem cells (both embryonic and iPS) to investigate the effects of chemicals on developmental processes Increased focus on genetic variation to understand susceptibilites - Toxicogenetics

NIEHS-NCATS-UNC DREAM Toxicogenetics Challenge An innovative crowdsourced computational challenge launched on June 11, 2013, and closed September 15, 2013. The NIEHS-NCATS-UNC team conducted the largest ever population- based in vitro cytotoxicity study: 1086 cell lines representing 9 distinct geographical populations. 179 drugs and environmental chemicals. https://www.synapse.org/#!Challenges:DREAM8 The challenge asked participants to build models based on data from the toxicogenetics project to predict: The toxic response of individuals to chemicals. How a particular population will respond to certain types of chemicals.

Benzene Inhalation Study with the DO Mouse Diversity outbred (J:DO) male mice selected from 175 breeding pairs Dose levels: 0, 1, 10, 100 ppm benzene, 28 days, 6 hr/day 600 mice total: 2 separate cohorts to assess reproducibility Endpoints for hematotoxicity and genetic damage % reticulocytes and micronucleated reticulocytes in bone marrow and blood Mouse Universal Genotyping Array (7.5K SNPs; MUGA) Mapping & Linkage analysis (QTLRel) Mice showed a 205-fold difference in susceptibility Associated with variable expression of a sulfotransferace detoxification enzyme French JE, et al. 2015. Environ Health Perspect. 123: 237 – 245. 13 Paules_2015

4/23/2017 Milestones of Tox21 Made all Tox21 Phase I and Phase II data collected to date public. Made chemical libraries available to investigators to expand the breadth of toxicological information. Identified artifacts in high throughput screening data that lead to false results. Made progress in data analysis and development of tools for prioritizing chemicals for more extensive testing using traditional methods. Exchanged assays and data with other organizations/efforts (e.g., EU Joint Research Centre, Health Canada, SEURAT, OpenTox). Worked with other NIEHS groups to evaluate Tox21 data for use by regulatory agencies.

Benefits of Tox21 Information and informatics tools from Tox21 will facilitate new drug discovery and promote translational research across NIH by eliminating substances with undesirable effects from further development. This federal multiagency collaboration is realizing a new vision for toxicological testing and contributing to improving public health.